我们想感谢肖恩·惠兰与拯救重组VSV变种的协助，以及技术援助的Didem的戈兹贝达和Ryan Chrenek为。这项工作是由霍华德休斯医学研究所（CLC），NS068012-01（KTB）。

1。使病毒从cDNA恢复的VSV的cDNA使用牛痘-T7系统10 <ol><li>前一天进行实验，分割成60毫米菜含有DMEM + 10％FBS BsrT7细胞。每道菜的种子2E6细胞。 BsrT7细胞来源于BHK21，或幼仓鼠肾细胞。 </li><li>暖用1mM镁和1mM氯化钙的PBS至37℃。 </li><li>获取一个小等分vTF7-3，表达T7聚合酶的牛痘病毒，和解冻至室温。 vTF7-3是一种传染性的牛痘病毒表达T7聚合酶11，并应使用仅在生物安全级别2遏制。这种表达系统是用来产生所需的转录从质粒转染在步骤1.9的最大水平。涡病毒在最大速度为30秒。为2分钟，在室温下在水浴中超声在台式超声波发生器。涡再次，持续30秒打破了病毒的团块。 </li><li>删除媒体从BSRT7细胞。 </li><li> 11.7微升vTF7-3到第600微升PBS与镁和钙，并添加到细胞的混合物。 </li><li>移动板成34℃培养箱中，用5％的CO 2。岩板轻轻每10分钟一次。 </li><li>经过45分钟后，开始转染。加入20μL脂质体转染法2000到1毫升DMEM和组合。等待5分钟。 </li><li>加入5.25微克pN时，PP 2.3微克，1.2微克复数，1微克pCAG RABV-G，和6μg的VSV基因组cDNA到500μlDMEM培养基。 PN，PP和pl质粒驱动下的T7启动子10的VSV病毒基因的表达，与根据经验确定的量的病毒的最佳救援。 </li><li>结合管和混合。等待15分钟，在室温下。 </li><li>删除PBS洗的盘子，用1.5毫升DMEM。 </li><li>吸冲洗介质。转染混合物板。移动板为34℃培养箱中培养。 </li><li>等待5个小时，然后取出介质。 </li><li>改变介质至4毫升DMEM + 2％FBS + 1个青霉素，链霉素+ 10 mM HEPES的pH值7.4。添加Penicillin - 链霉素，以防止污染，和HEPES，以维持pH的媒体，是非常重要的。 FBS含量可降至2％，被破坏的细胞在短短几天的病毒，因此不需要10％FBS。 </li><li>将板为34℃培养箱中培养48-72小时。 </li><li>收藏报错媒体在第3和转染后6天，并立即通过一个0.20微米的过滤器的过滤器的注射器。这个尺寸的过滤器除去的牛痘病毒，但不VSV。 </li><li>既然我们要提供RABV-G的病毒包膜，和基因组不编码RABV-G基因，它必须不断地供给反要做到这一点，使一个单独的盘的:293T TVA800（TVA800）细胞。这些细胞是组成型表达TVA受体，允许ENVA介导的病毒感染12。在80％汇合时，更改只媒体到DMEM，然后转染的细胞，用5μg的质粒编码G蛋白（ 即 pCAG RABV-G），每块板。我们用PE我转染试剂，聚阳离子聚合物，它是便宜得多的可比脂质为基础的方法。然而，脂质体转染法也可以用来有效地用于此目的。最佳PEI:DNA的比例需要根据经验来确定。在另一项实验中， 即 pCAG GFP的荧光记者。我们使用10微升PEI:4微克DNA。 </li><li>应用从步骤16到转染的板TVA800细胞上清液。 </li><li>观察这个板块的翌日病毒表达荧光的荧光的证据。首次观察到病毒的荧光作为一个单一的单元格中，并且随着时间的推移（ 即 图3）逐渐传播。该病毒在几个小时内蔓延。 </li></ol> 2。 VSV的通道和浓度<ol><li> TVA800细胞分割成含DMEM + 10％FBS这样，你将有4个10厘米〜80％汇合第二天板。由于他们的高转录293T为基础的电池的工作原理sfectability TVA-表达细胞，并且使用增强的速度，在盘的细胞被感染。其他高效转染的细胞系可以达到同样目的。 </li><li>在80％融合，改变媒体的DMEM，然后用5μg您要使用的（ 即 pCAG RABV-G）每板的G蛋白的质粒转染的细胞。我们使用PEI，但FUGENE的/脂质体的工作为好。 </li><li>等待一天后，转糖蛋白的表达/表面积累。更改媒体（5毫升/板），然后到DMEM + 10％FBS的感染与rVSV病毒（A / RG），在0.01 &lt;X &lt;0.1的感染复数（MOI），。 </li><li>检查24小时后的感染量。你应该看到感染的细胞（由GFP荧光标识），一些与感染的细胞（ 即 图3）的周边补丁。通常情况下，这是唯一值得收集上清液，如果你看&gt; 50％的细胞受到感染，否则病毒滴度obtained的浓度将是最理想的。 </li><li>如果&gt; 50％，收集5毫升，媒体/板，并更换新鲜的培养基中，用5毫升的。如果太少的细胞被感染，另一个等待12-24小时，并再次检查。冻结收集的上清液在-80℃下</li><li>收集每24小时后3天，4天，总。 </li><li>总之，如果你开始有4个板块，你应该有媒体每天20毫升 - 所以80毫升总，或价值2超速离心管的上清液。解冻后的上清液在37℃下，使用超过0.45微米的过滤器在冰上。等分试样36毫升的病毒到Beckman离心机管。 </li><li>浓缩上清液（21,000 K的一个SW28转子（= 80,000 xg离心）90分钟）在4℃下到含有10％的漂白剂的烧杯中，滗析出上清液，同时管被反转，使用抽吸器除去尽可能多的媒体从侧管尽可能。 </li><li>在覆盖。动摇病毒管在4℃下1小时。在阿布一个标准的振动筛吨120 rpm是足够的。 </li><li>轻轻捣碎沉淀，剩余的介质，轻轻上下吹打30次。要小心，不要引入气泡。剩余的媒体数量应约30微升。此卷为多管分装，以避免多次冻融循环对任何特定的股票，因为这可能会导致在病毒滴度下降。可以冻结这些等分试样在-80℃下</li><li>滴度的病毒。在大约2天感染后滴定是最优的。最初的感染可在一小时内就可以观察到，但你会得到一个代表名额不足的滴度，如果你等待的只有1天。为了获得的病毒滴度，进行限制性稀释浓缩的病毒实验到在24孔板中的合适的细胞系（293T细胞中很好地工作），稀释10倍的病毒从第1至1:1,000,000。我们得到的效价通常是在10 8 -10 10焦点形成单位（FFU）/毫升的范围内。 </li></ol> 3。维尔我们注射<ol><li>我们使用的小鼠之间，通常在6-10周龄，这些实验。任何年龄建立后，电路就足够了。我们会选择从视网膜神经节细胞（RGC）的传输到爆的无长突细胞（SACS）为例。本实验中，在图2中的框图，需要一个三元组的转基因动物，只有一个单一的注射的病毒。 （所有程序批准的IA​​CUC在哈佛医学院，按照与机构的指导方针）。 </li><li>首先，需要以产生适当的动物。我们的目标是有TVA表达的细胞类型中的一个人希望映射到的病毒（ 如腺病毒相关病毒，AAV）的装置，通过，或通过转基因的等位基因。 Cre表达的细胞类型特异性通常是通过以下方式获得。在这里，我们也越过TVA在条件表达式中等位基因7胆碱乙酰转移酶（CHAT）中铁快运等位基因13，TVA表示在一个的Cre表达历史记录（诊所）与所有细胞。我们也越过一个条件表达式tdTomato的等位基因（R26TdT）的TVA-细胞可视化。 </li><li>注入位置的坐标为需要确定。这些坐标的利益，可以发现在脑图谱， 即富兰克林和Paxinos鼠脑图谱14。这些坐标需要注意的小鼠颅骨相对于一个给定的地标。我们使用前囟门，但任何坐标系是足够的。 </li><li>准备立体定位仪。打开的微量注射器控制器上，并移动的柱塞和电极座入到位。然后回到与矿物油填充的注射针，提供一个接口，与病毒。把唱针，然后到柱塞上，确保没有气泡留在针。 </li><li>解冻的病毒，并将其放置在冰上，以防止病毒滴度下降。将微量注射器"退出"，将管的病毒，是contacti在毛细管上的微量注射器。 RABV-G假rVSV（A / RG）的实验中提取必要的量。 </li><li>动物被给予手术前剂量的丁丙诺啡（0.05-0.1毫克/公斤），在手术开始之前。对于麻醉动物，无论是异氟醚吸入，或腹膜内注射氯胺酮（40-80毫克/千克）和甲苯噻嗪（5-10毫克/千克）的混合物是足够的。麻醉剂的选择是依赖于用户。脚趾夹，然后执行，以确保动物是完全麻醉，如果动物没有反应，你可以继续程序。使用这些药物之前，请务必以获得适当的许可。 </li><li>动物被放置在加热垫，支撑一个可移动的支架上，用于定位鼠标。动物仍然存在在整个外科手术的持续时间。应用眼药膏，以防止角膜干燥，而动物麻醉。 </li><li>鼠标的头部，然后需要稳定的立体一个pparatus。使用耳杆的立体定位仪上，以确保头部彻底担保，使得头部与地面平行，和不旋转横向。一旦这项工作完成，调整咬栏，以帮助稳定头。 </li><li>动物的皮毛被剃光头顶上在该地区的切口。此区域，然后用乙醇洗涤三次，接着用碘由三次。一个切口，然后用手术刀在皮肤上，露出了头骨。 </li><li>鼠标和注射设置一旦准备好了，前囟门必须位于。这是该领域中的头部的中心，在矢状面和两个冠状缝满足。一旦此区位于，立体定位仪上的刻度盘可以被用于调节到适当的坐标的微量注射器。在这里，我们注入外侧膝状体（LGN），使用坐标A / P，L / M 2，D / V 2.75 -2.46从囟。 </li><li>一旦坐标已经被拨号时，钻头必须进行汇编。将钻头钻钻，然后打开。一个小的孔应钻成的站点确定为注射部位。很温柔的演习中，以不引起脑实质的损害。当骨非常薄，硬脑膜血管变得清晰起来。在这一点上，仔细穿孔开颅的边缘与一个小的（30表压）的针和罚款（＃5）的钳子，以除去剩余的骨。 </li><li>调整毛细管孔钻的大脑表面，只是背的位置。针头应该是足够清晰的穿透硬脑膜轻松的。然后针降低到所需的深度，并开始注射。我们注入病毒在100升/分钟的速率。 </li><li>这一点的注射程序需要大致10分钟。注射后，针保持在大脑中约七分钟。这是至关重要的，因为它允许从注射部位的病毒扩散。快速去除针形，麟蹄ction网站的查询结果在降低在注射部位的感染效率，并沿针道感染。 </li><li>将针从注射部位，非常缓慢地升至跨度超过大约两分钟。这又是打算沿针道，以减少感染。 </li><li>一旦移除，然后缝合皮肤。动物连续监测，直到他们从麻醉中恢复过来。丁丙诺啡（0.05-0.1毫克/公斤），每12小时2天。 </li></ol> 4。收获组织/组织准备<ol><li>对于收获感兴趣的组织中，最佳时间取决于实验的目标。一般来说，VSV跨突触传播可以先观察到小于24小时后喷射。然而，等待更长的时间，会导致更多的传播。 </li><li>要收获的视网膜，动物是第一个安乐死，使用吸入二氧化碳和颈椎脱位执行。眼球然后除去，放入的管浓度含在PBS中的4％多聚甲醛在室温下1小时。 </li><li>在眼球上，然后放置到陪替氏培养皿，用PBS。视网膜解剖远离其他组织。 </li><li>对于整装分析，视网膜被切断，平躺着，放到载玻片上，然后安装延长黄金的安装媒体，并覆盖着一个零厚度的盖玻片。在视网膜上的压缩力，以尽量减少载玻片和盖玻片之间放置硅衬垫。对于截面分析，被放置在视网膜视网膜淹没在PBS中，直到30％的蔗糖。然后放入50％的OCT的混合物和50％30％蔗糖溶液中3小时。然后闪烁冻结，并保持在-80℃，直到准备切割。 </li></ol> 5。代表性的成果应该能够从cDNA拯救的病毒感染TVA800细胞，但不是293T细胞（ 图3）。供应RABV-G可以感染多种细胞吨YPES，包括293T。然而，由于基因组中的基因组编码的A / RG，细胞间传播发生在TVA-表达细胞中的高得多的速率。 一旦病毒浓缩，典型的10 8 FFU / ml和10 10 FFU / ml的滴度范围之间。用于在体内这些滴度是足够的。 到小鼠在LGN注射过程中，我们不区分背LGN（背外膝体）从腹侧LGN（vLGN）的，通常都被感染。因此，多个RGC的类型成为标记（ 图4），包括那些项目的vLGN，如melanopsin的视网膜神经节细胞（ 图4D）和ON-DSGCs（ 图4E），以及那些项目的后背外侧膝状体，如小乔木视网膜神经节细胞（ 图4C）和ON-OFF-DSGCs（ 图4F）。虽然不止一种类型的RGC传播病毒到物质误用诊所，在其他地方（贝尔等人 ，在审查），在这里我们只关注研究ON-OFF-DSGCs的连接到物质误用诊所。 当我们注入小鼠的基因型cTVA（+）/聊天中铁快运（ - ），在没有TVA应表示，只有视网膜神经节细胞标记（ 即 图4）。这是由于由视网膜神经节细胞的RABV-G假型病毒的初始摄取，而没有扩散发生。无视网膜神经节细胞被感染的LGN感染的rVSV，（A / RG）病毒RABV-G不假，由于这些病毒颗粒无法横向长这些视网膜神经节细胞轴突。但是，当注射cTVA（+）/（+）（和R26TdT（+））ChATCre小鼠成的LGN，病毒扩散并发生，只有红色的细胞，该细胞表达的Cre记者（ 即 图5）。这些细胞也共同标签与抗ChAT抗体，仅在两个ChAT的薄层和分层作为评估中心服务（ 图5B&#39;，C&#39;），确认其身分。每DSGC病毒标记的物质误用诊所的数量从一到九之间。 图1跨突触。我们的逆行追踪系统相比最初的开发方法。 （A）（i）在开发由威克沙姆和他的同事的方法，"起动细胞转染与这三种基因的TVA受体，允许感染，特别是转染细胞; RABV-G，用于补充RABV本身有G基因删除;和红色荧光蛋白，以确定转染细胞。 （ 二 ）的RABV假的A / RG蛋白感染TVA-细胞，使它们黄色的。 （ 三 ）发生逆行跨突触传递到上游的神经元。 （B）（ⅰ），在我们的方法中，TVA-表达细胞定义的基因，与一个有条件的红色荧光蛋白A，并标示为英寸（ⅱ）"起动机细胞"是指由一个VSV病毒基因组中的基因编码的A / RG感染。这些初始细胞不表达TVA受体。 （ 三 ）逆行跨突触传递只有当这些细胞发生TVA表达神经元突触输入的首发神经元上。 <a href="http://www.jove.com/files/ftp_upload/4253/4253fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" fo:content-width="4.6in" fo:src="/files/ftp_upload/4253/4253fig2highres.jpg" src="/files/ftp_upload/4253/4253fig2.jpg" /> 图2的实验步骤的示意图。 （A）首先，病毒从cDNA救出。然后传代RABV-G假型化，并浓缩和滴定。此病毒，然后注射到大脑，在那里它被允许孵育期望的时间段内。在此之后时间，眼睛被收获，和视网膜的解剖和分析。 （B）RABV-G假型病毒的原理图。这是必要的视网膜神经节细胞从大脑注射感染。 RABV-G基因转染组织培养细胞中表达的TVA受体。然后，这些细胞的的A / RG（rVSV）的病毒感染。释放的病毒粒子，将有RABV-G在病毒包膜，但不会有RABV-G基因的病毒基因组中。 （C）的示意性的鼠标穿过必要三重的转基因动物。有条件的TVA和tdTomato等位基因的交叉到Cre重组酶的选择，比如，TVA和tdTomato都表示只有在细胞中Cre表达历史与驱动程序。在这个例子中，我们的聊天中铁快运。 （D）的视网膜感染的示意图，以便追踪DSGC-SAC电路。 ChAT的细胞作出明示TVA和tdTomato的，如在（C）所示。从LGN在视网膜神经节细胞被感染（B）中产生的病毒的fection。由于病毒表达绿色荧光蛋白，，这些视网膜神经节细胞将是绿色。然后，该病毒会传播只TVA表达ChAT的无长突细胞，如果它们连接到标记RGC。这些细胞将是红色和绿色或黄色的。 <a href="http://www.jove.com/files/ftp_upload/4253/4253fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/4253/4253fig3highres.jpg" src="/files/ftp_upload/4253/4253fig3.jpg" /> 图3。的rVSV病毒（A / RG）上293T和TVA800细胞的行为。这些细胞被感染低:滴度rVSV（A / RG）与RABV-G假型。该病毒之间的利差TVA-细胞在几个小时内，感染后6小时（HPI），感染后2天（DPI）所示。然而，293T细胞，不表达TVA受体的蔓延，虽然我吨确实发生，发生慢得多。比例尺= 100微米。 <img alt="图4" fo:content-width="4in" fo:src="/files/ftp_upload/4253/4253fig4highres.jpg" src="/files/ftp_upload/4253/4253fig4.jpg" /> 图4代表非TVA表达小鼠的视网膜神经节细胞的感染。动物注射到LGN的rVSV（A / RG）RABV-G假型病毒，组织收获2 DPI。 （A）的稀疏（视网膜神经节细胞由黄色箭头表示），或（B）感染可以得到致密的，这取决于病毒的滴度和注射质量。根据形态，包括（C）小乔木视网膜神经节细胞（D）1型黑视素的视网膜神经节细胞（E）ON-DSGCs"，和（F）ON-OFF-DSGCs，其中包括许多不同类型的视网膜神经节细胞可以识别。比例尺= 50微米。 <img alt="图5"FO:内容的宽度="4英寸"佛:SRC ="/ files/ftp_upload/4253/4253fig5highres.jpg"SRC ="/ files/ftp_upload/4253/4253fig5.jpg"/&gt; 图5。rVSV（A / RG），从ON-OFF-DSGCs的物质误用诊所传输遵循预期的模式。 （A）ON-OFF-DSGCs（白色箭头与绿色填充）传播病毒到多个气囊（黄色箭头）。 （A&#39;）。不是所有绿色细胞的Cre记者DSGC合作标签，这表明它们是表达TVA-评估中心服务。 （BC），DSGC（绿色填充白色箭头）及特别评估中心（黄色箭头）在适当的分层聊天层的视网膜内丛状层（IPL）。细胞的病毒传送ON和OFF-物质误用诊所。面板中的B&#39;，C&#39;的数字表明IPL的纹层。 DSGC胞体的位置仅由箭头表示，但面板 C中未示出，以反白显示的SAC。比例尺= 50微米。

<ol>
	<li>Wickersham, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+restriction+of+transsynaptic+tracing+from+single,+genetically+targeted+neurons.">Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons.</a> Neuron. 53, 639-647 (2007).</li><li>Marshel, J. H., Mori, T., Nielsen, K. J., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+single+neuronal+networks+for+gene+expression+and+cell+labeling+in+vivo.">Targeting single neuronal networks for gene expression and cell labeling in vivo.</a> Neuron. 67, 562-574 (2010).</li><li>Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+circuit+tracing+in+vivo+through+Cre-dependent+targeting+and+complementation+of+modified+rabies+virus.">Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 21848-21853 (2010).</li><li>Wickersham, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+restriction+of+transsynaptic+tracing+from+single,+genetically+targeted+neurons.">Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons.</a> Neuron. 53, 639-647 (2007).</li><li>Yonehara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+asymmetric+reorganization+of+inhibition+establishes+a+motion-sensitive+circuit.">Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit.</a> Nature. 469, 407-410 (2011).</li><li>Stepien, A. E., Tripodi, M., Arber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+rabies+virus+reveals+premotor+network+organization+and+synaptic+specificity+of+cholinergic+partition+cells.">Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells.</a> Neuron. 68, 456-472 (2010).</li><li>Beier, K. T., Samson, M. E. S., Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+expression+of+the+TVA+receptor+allows+clonal+analysis+of+descendents+from+Cre-expressing+progenitor+cells.">Conditional expression of the TVA receptor allows clonal analysis of descendents from Cre-expressing progenitor cells.</a> Dev. Biol. 353, 309-320 (2011).</li><li>Seidler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cre-loxP-based+mouse+model+for+conditional+somatic+gene+expression+and+knockdown+in+vivo+by+using+avian+retroviral+vectors.">A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 10137-10142 (2008).</li><li>Beier, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterograde+or+retrograde+transsynaptic+labeling+of+CNS+neurons+with+vesicular+stomatitis+virus+vectors.">Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 15414-15419 (2011).</li><li>Whelan, S. P., Ball, L. A., Barr, J. N., Wertz, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+recovery+of+infectious+vesicular+stomatitis+virus+entirely+from+cDNA+clones.">Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 8388-8392 (1995).</li><li>Fuerst, T. R., Niles, E. G., Studier, F. W., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+Transient-Expression+System+Based+on+Recombinant+Vaccinia+Virus+That+Synthesizes+Bacteriophage+T7+RNA+Polymerase.">Eukaryotic Transient-Expression System Based on Recombinant Vaccinia Virus That Synthesizes Bacteriophage T7 RNA Polymerase.</a> PNAS. 83, 8122-8126 (1986).</li><li>Young, J. A., Bates, P., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+chicken+gene+that+confers+susceptibility+to+infection+by+subgroup+A+avian+leukosis+and+sarcoma+viruses.">Isolation of a chicken gene that confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses.</a> J. Virol. 67, 1811-1816 (1993).</li><li>Madisen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain.">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nat Neurosci. 13, 133-140 (2010).</li><li>Franklin, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>van den Pol, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+strategies+for+studying+the+brain,+including+a+replication-restricted+self-amplifying+delta-G+vesicular+stomatis+virus+that+rapidly+expresses+transgenes+in+brain+and+can+generate+a+multicolor+golgi-like+expression.">Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression.</a> J. Comp. Neurol. 516, 456-481 (2009).</li></ol>

没有利益冲突的声明。

使用病毒来研究神经回路是一个相对较高的吞吐量，连接神经元的分析方法。但是，生成，VSV和RABV病毒颗粒是不平凡的。虽然提供了上面列出的用于从cDNA拯救病毒的协议，它仍然是一个低概率事件。水平的每一个的N，P和L的质粒中需要被精确地调整，需要做的工作，以确保病毒救援和许多试验和复制。将一个完整的VSV基因组RNA的核苷酸颗粒的形成是一个低概率事件，因此可能需要多次尝试。这?...

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Tissue Culture</td>
		</tr>
		<tr>
			<td>Baby Hamster Kidney (BSRT7) cells</td>
			<td>available upon request</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>vaccinia (vTF7-3)</td>
			<td>available upon request</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>pN, pP, pl plasmids</td>
			<td>available upon request</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>Open Biosystems</td>
			<td>HCL4517</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>60 mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>75 cm2 Rectangular Canted Neck Cell Culture Flask with Vent Cap</td>
			<td>Corning</td>
			<td>430641</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Media : DMEM (Dulbecco's Modified Eagle Medium)</td>
			<td>Invitrogen</td>
			<td>12491-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 M HEPES pH 7.4</td>
			<td>Gibo</td>
			<td>15630-080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS: Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PKS</td>
			<td>Invitrogen</td>
			<td>15140-163</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2,000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe: 5 ml Luer-Lock syringe</td>
			<td>Sigma</td>
			<td>Z248010-1PAK</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>Nalgene</td>
			<td>190-2520</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PEI: High Potency Linear PEI</td>
			<td>Polysciences</td>
			<td>23966</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Viral Centrifugation</td>
		</tr>
		<tr>
			<td>Corning 150 ml Tube Top Vacuum Filter System, 0.45 &mu;m Pore</td>
			<td>Corning</td>
			<td>430314</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thinwall, Ultra-Clear, 38.5 ml, 25 x 89 mm ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>344058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td>optima XL-80K</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SW28 Ultracentrifuge rotor</td>
			<td>Beckman-Coulter</td>
			<td>342207</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Mouse Injection</td>
		</tr>
		<tr>
			<td>Capillary micropipets</td>
			<td>Drummond</td>
			<td>5-000-2005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereotax</td>
			<td>Narishige</td>
			<td>SR-5M</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>SM-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ump injector</td>
			<td>World Precision Instruments</td>
			<td>Sys-Micro4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Four channel microcontroller</td>
			<td>World Precision Instruments</td>
			<td>UMP3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>M.TXB Bench Motor with C.EMX-1 Dial Control, 115 Volt</td>
			<td>Foredom</td>
			<td>M.TXB-EM</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>H.10 Handpiece, Quick Change</td>
			<td>Foredom</td>
			<td>H.10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Step Drill, 0.5 mm</td>
			<td>Foredom</td>
			<td>A-58005P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microelectrode holder</td>
			<td>World Precision Instruments</td>
			<td>MEH2S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>995-2949</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Henry Schein</td>
			<td>4015809TV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Henry Schein</td>
			<td>1118217</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Becton-Dickinson</td>
			<td>309628</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 gauge injection needle</td>
			<td>Becton-Dickinson</td>
			<td>305106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protective Ophthalmic Ointment</td>
			<td>Doctors Foster and Smith</td>
			<td>9N-014748</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>493511</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Sigma</td>
			<td>PVP1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Surgery and Dissection tools</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>91402-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Fine Science Tools</td>
			<td>10015-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Robbins Instruments</td>
			<td>20.SK640</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Dissection and antibody staining</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Antibodies</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td>millipore</td>
			<td>AB144P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-gfp</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey anti-chicken Dylight 488</td>
			<td>Jackson immunoresearch</td>
			<td>703-545-155</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey anti-chicken Alexa Fluor 647</td>
			<td>Jackson immunoresearch</td>
			<td>705-605-147</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Superfrost plus microscope slides</td>
			<td>Fisher</td>
			<td>12-550-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cover glass 22 x 22, 0 thickness</td>
			<td>Electron Microscopy Sciences</td>
			<td>72198-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>Rogers Corp</td>
			<td>HT-6220</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

viral tracing of genetically defined neural circuitry

古典方法研究神经回路是相当低的吞吐量。病毒跨突触，特别是伪狂犬（PRV）和狂犬病毒（RABV），，最近水泡性口炎病毒（VSV），学习电路，正变得越来越流行。这些高通量的方法使用顺行或逆行方向的神经元之间传递的病毒。 近日，RABV突触逆行追踪。 （ 图1A）。在这种方法中，糖蛋白（G）的基因被删除从病毒基因组中，仅在目标神经元和补给。感染特异性达到代的嵌合G，ASLV-阿糖蛋白和胞质域的RABV-G（A / RG）的胞外结构域组成，正常RABV-G 1。特别是感染这种嵌合G TVA受体1的表达。该基因编码TVA已经Delivered通过各种方法2-8。 RABV-G感染的TVA表达神经元，RABV可以传输到其他突触连接的神经元在逆行方向的性质，这是交付的TVA受体的G。这种技术标签相对大量的输入（5-10％）2到一个定义的细胞类型，提供了一个采样到一个定义的起动器的细胞类型中的所有的输入。 我们最近修改了这个技术的使用VSV作为一个跨突触示踪9。 VSV有几个好处，包括快速的基因表达。在这里，我们详细介绍了新的病毒跟踪系统，使用VSV有用的探测微型电路与更高的分辨率。在原来的出版策略，由威克沙姆等。4和贝尔等人。9许可证标记神经元的任何项目上最初感染TVA-表达细胞，在这里VSV被设计仅传输到电视A-细胞（ 图1B）。该病毒是第一个假RABV-G允许感染下游TVA表达神经元的神经元。这个第一群细胞感染后，病毒发布只能感染TVA-表达细胞。由于跨突触的病毒传播是有限的TVA-细胞的自定义类型的细胞连接的情况下，存在可以被探索和高分辨率。这些实验是在图2中所示的实验流程图。在这里，我们展示一个模型电路，在小鼠视网膜的方向选择性。我们考察爆无长突细胞（SACS）的连通性视网膜神经节细胞（RGC）。

Watch this Scientific Journal Video about Viral Tracing of Genetically Defined Neural Circuitry at JoVE.com

Viral Tracing of Genetically Defined Neural Circuitry

跟踪突触连接的神经元的方法。我们使用TVA特异性的上游电池，探测是否感兴趣的接收的细胞群体突触输入从基因定义的细胞类型。

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

viral-tracing-of-genetically-defined-neural-circuitry

Research

JoVE Journal

Neuroscience

18.4K 次观看.  Harvard Medical School. 跟踪突触连接的神经元的方法。我们使用TVA特异性的上游电池，探测是否感兴趣的接收的细胞群体突触输入从基因定义的细胞类型。

视频: Viral Tracing of Genetically Defined Neural Circuitry

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

科研

神经科学

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

新生大鼠于A气味偏侧学习模型解剖神经回路托换记忆的形成

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

有条件的遗传跨突触标记在胚胎小鼠脑

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

小鼠膈运动神经元逆行神经解剖学追踪的研究

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...